Hybrid Superparamagnetic Iron Oxide Nanoparticles And Polyethylenimine As A Magnetoplex For Gene Transfection

ABSTRACT

Disclosed are the nanoparticle and the method for the same, and the preparing method includes steps of mixing polyethylenimine (PEI) with the poly(acrylic acid)-bound iron oxide (PAAIO) to form a PEI-PAAIO polyelectrolyte complex (PEC) and mixing the PEI-PAAIO PEC with genetic material such as plasmid DNA to form the PEI-PAAIO/pDNA magnetic nanoparticle. The PEI-PAAIO/pDNA magnetoplex is highly water dispersible and suitable for long term storage, shows superparamagnetism, low cytotoxicity, high stability and nice transfection efficiency, and thus the PEI-PAAIO PEC can replace PEI as a non-viral gene vector.

CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY

The application claims the benefit of Taiwan Patent Application No.099144853, filed on Dec. 20, 2010, in the Taiwan Intellectual PropertyOffice, the disclosures of which are incorporated herein in theirentirety by reference.

FIELD OF THE INVENTION

The present invention relates to nanoparticles. In particular, thepresent invention relates to the nanoparticles made with hybridsuperparamagnetic iron oxide and polyethylenimine. The genetic materialscarried by the nanoparticles can be transfected into cells, and suchnanoparticles can be applied in clinic medicine.

BACKGROUND OF THE INVENTION

Transfection or transformation is referred to the technology fortransporting the genetic materials into cells or organisms. Forinstance, an electric field is performed on the plasmid DNA, and theplasmid DNA then is transported into cells to express its biologicalfunctions or to be translated as specific proteins. Alternatively,liposome with positive charge is mixed with negative charge DNA to formcomplex, and DNA is delivered to cytoplasm by the fusion of the complexwith cell membrane or endocytosis.

U.S. Pat. No. 6,846,809 discloses a DNA vector for DNA delivery, whereinnucleic acid and polycation are mixed to form a liquid transfectioncomposition, which directly contacts with cancer cells or tissues havingcancer cells, so that nucleic acid is delivered to cancer cells toinhibit their growth. However, since no external electric field ormagnetic field is performed on the liquid transfection composition, thetransfection efficiency is not high.

In recent years, the magnetic transfection technology is developed,which integrates the genetic materials such as DNA, small interferingRNA (siRNA) and so on with magnetic nanoparticles to transport thegenetic materials using a magnetic field. For instance, US PatentPublication No. 2008/0075701 discloses a composition for magnetofection,which envelopes the magnetic nanoparticles (MNPs) and the geneticmaterials inside the hydrophilic vector (liposome) to form the sphericalvehicle. At first, the MNPs are coated with surfactant (organic acid),and the excess surfactant is removed with ultrasonication. The MNPs thenare enveloped inside the liposome, but surfactant is not envelopedthereinside. Subsequently, the genetic materials are added to the fluidcontaining liposome-enveloped MNPs, and the genetic materials pass intothe liposome via the lipid bilayer structure to form the end product,the spherical vehicle. However, the processing steps of the MNPs in US2008/0075701 are too complicated, and the genetic materials need to passthrough the lipid bilayer structure to be enveloped with the liposome.Thus, it will reduce the probability that the nanoparticles and magneticmaterials are enveloped inside the liposome at the same time.

The MNP based on iron oxide nanoparticles can be degraded inphysiological conditions over a time period of a month suspiciouslycorresponding to ferritin synthesis (Briley-Saebo et al., 2004). Sincethe internalized iron oxide nanoparticles are biotransformed, thus,there would be no safety concern using MNPs as a drug delivery system.

SUMMARY OF THE INVENTION

For overcoming the safety problem of the MNPs in the currenttechnologies, the MNPs (or named magnetoplexes) of the present inventionare highly water dispensable and can be longtime reserved, and haveproperties of superparamagnetism, low cytotoxicity, high stability andexcellent transfection efficiency. The MNPs can replace polyethylenimine(PEI) to be the non-viral gene vector.

The present invention provides a method for preparing a nanoparticle,including steps of: reacting a PEI with a polyacrylic acid-bound ironoxide (PAAIO) to obtain a polyelectrolyte complex (PEC); andencapsulating (or incorporating) a genetic material with the PEC to formthe nanoparticle. The above preparation method of the present inventionis simple and efficient with a high yield.

The present invention further provides a nanoparticle afforded by theaforementioned preparation method, and the nanoparticle includes: thePEC containing the PEI and the PAAIO configured on the PEI; and agenetic material coupled to the PEI.

The present invention further provides a nanoparticle, including: a PECcontaining a positive charge molecule and a magnetic particle configuredon the positive charge molecule; and a genetic material coupled to thepositive charge molecule.

The positive charge molecule includes but not limit to PEI, andPEGlayted PEI also can be applicable in the present invention. ThePEGlayed PEI is referred to that polyethylene glycol (PEG) polymerchains are covalently attached to the PEI. The magnetic particleincludes but not limit to metal particles. The metal particles includebut not limit to gold nanoparticles, silver nanoparticles and ironnanoparticles, and the genetic material can be DNA, RNA, complementaryDNA, small interfering RNA (siRNA), micro RNA and so on.

The above objectives and advantages of the present invention will becomemore readily apparent to those ordinarily skilled in the art afterreviewing the following detailed descriptions and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts a diagram showing the PEI-PAAIO/pDNAmagnetoplex of the present invention.

FIGS. 2( a) and 2(b) respectively illustrate the diagrams showing (a)hydrodynamic particle size and (b) zeta potential using two weightratios between PAAIO and PEI in the preparations two PEI-PAAIOcomplexes.

FIG. 3( a) illustrates the diagrams showing FTIR spectrum of PEI, PAAIOand PEI-PAAIO complexes.

FIGS. 3( b) and 3(c) illustrates the diagrams showing ESCA spectra of(b) PAAIO and (c) PEI-PAAIO complexes.

FIG. 3( d) illustrates the diagram showing SQUID magnetization curves asa function of field at 25° C. for PAAIO and PEI-PAAIO complexes.

FIGS. 4( a) and 4(b) respectively illustrate the diagrams showing (a)particle size and (b) zeta potential of PEI-PAAIO/pDNA magnetoplex atvarious N/P ratios.

FIGS. 5( a) and 5(b) respectively illustrate the diagrams showing (a)cytotoxicity induced by PEI-PAAIO against HEK 293T cells at variousconcentration and (b) cytotoxicity induced by PEI-PAAIO/pDNAmagnetoplexes at different N/P ratios with or without a magnetic field.

FIGS. 6( a) and 6(b) respectively illustrate the diagrams showingpEGFP-C1 expression in HEK 293T cells exposed to PEI-PAAIO/pDNAmagnetoplexes (a) without 10% FBS and (b) with 10% FBS in the absence orpresence of a magnetic field for 4 hours incubation followed by 72 hourspost-incubation.

FIGS. 7( a) and 7(b) respectively illustrate the diagrams showingluciferase activity in HEK 293T cells exposed to PEI-PAAIO/pDNAmagnetoplexes (a) without 10% FBS and (b) with 10% FBS in the absence orpresence of a magnetic field for 4 hours incubation followed by 72 hourspost-incubation.

FIGS. 8( a) and 8(b) respectively illustrates the diagrams showing theHEK 293T cell-internalized iron oxides in PEI-PAAIO/pDNA magnetoplexesby ICP-OES (a) without 10% FBS and (b) with 10% FBS in the absence orpresence of a magnetic field for 4 hours incubation.

FIGS. 9( a) and 9(b) respectively illustrate the diagrams showing theluciferase activity in U87 cells exposed to PEI-PAAIO/pDNA magnetoplexes(a) without 10% FBS and (b) with 10% FBS in the absence or presence of amagnetic field for 4 hours incubation followed by 72 hourspost-incubation.

FIGS. 9( c) and 9(d) respectively illustrate the diagrams showing theU87 cell-internalized iron oxides by ICP-OES (c) without 10% FBS and (d)with 10% FBS in the absence or presence of a magnetic field for 4 hoursincubation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more specifically withreference to the following Embodiments. It is to be noted that thefollowing descriptions of preferred Embodiments of this invention arepresented herein for purpose of illustration and description only; it isnot intended to be exhaustive or to be limited to the precise formdisclosed.

The magnetic nanoparticle (MNP) of the present invention includes metaloxide, carboxylic molecules, positive charge molecules, and geneticmaterials. The genetic materials include but not limit to the nucleicacid molecule such as DNA, RNA, complementary DNA (cDNA), smallinterfering RNA (siRNA), micro RNA and so on. The metal oxide can bebound with an acidic molecule, and the genetic material iselectrostatically coupled to the positive charge molecule. In thepresent invention, PEI and PEGlayted PEI can be used as the positivecharge molecule, in which PEGlayed PEI is referred to that polyethyleneglycol (PEG) polymer chains are covalently attached to the PEI.

Please refer to FIG. 1, which schematically depicts a diagram showingthe PEI-PAAIO/pDNA magnetic nanoparticle (or magnetoplex) of the presentinvention. PEI-PAAIO/pDNA magnetoplex includes iron oxide 1,polyethylenimine (PEI) 2 and plasmid DNA (abbreviated as pPDA) 3.Poly(acrylic acid) (PAA) 4 is bound with Fe₃O₄ 1 to form the PAA-boundiron oxide (PAAIO), which is obtained via a one-step reaction of Fe₃O₄coated with PAA 4. The negative charge pDNA 3 is electrically coupled tothe positive charge PEI 2 to form the PEI-PAAIO/pDNA magnetoplex. ThePEI-PAAIO/pDNA magnetoplex formed by coupling PEI and PAAIO can be thenon-viral gene vector. The metal particle being the core ofPEI-PAAIO/pDNA magnetoplex is water soluble, and include but not limitto gold nanoparticle, silver nanoparticle and iron nanoparticle.

EXPERIMENTS 1. Major Reagents

Iron(III) chloride, anhydrous (FeCl₃), and poly(acrylic acid) (PAA, Mw2,000 g/mol) were obtained from TCI (Tokyo, Japan). Polyethylenimine(PEI, branched, Mw 25,000 g/mol) was acquired from Polyscience(Warrington, Pa., USA). Potassium hexacyanoferrate (II) trihydrate waspurchased from Showa (Tokyo, Japan). The reporter gene pEGFP-C1 waspurchased from Clontech (Palo Alto, Calif., USA) and pGL3-control andits luciferase assay kit with reporter lysis buffer was purchased fromPromega (Madison, Wis., USA). The aforementioned plasmid DNAs (pDNA),pEGFP-C1 and pGL3-control, were propagated in a chemically competentEscherichia coli stain DH5α (GibcoBRL, Gaitherbury, Md., USA), andpurified by Viogene Plasmids Maxi kit (Viogene, Sunnyvale, Calif., USA).

2. Synthesis of PAAIO and PEI Coated PAAIO (PEI-PAAIO)

PAAIO was synthesized via a one-step reaction of Fe₃O₄ coated with PAAaccording to U.S. patent application Ser. No. 12/694,599. PEI coatedPAAIO (PEI-PAAIO) was prepared by mixing PEI and PAAIO at a stockconcentration of 1 mg/mL in double deionized (DD) water. The totalvolume of 15 mL of PEI and PAAIO prepared at two weight ratios of 1/1 or1/2 was used for hydrodynamic particle size and zeta potential tests.The PEI-PAAIO nanoparticles were ultrasonicated for 5 minutes. Theunbound PEI was removed by placing a permanent magnet (Nd—Fe—B of 6000Gauss (G)) near the test tube and the supernatant solution was carefullywithdrawn. The magnetic-attracted PEI-PAAIO nanoparticles wereresuspended in 15 mL DD water and centrifuged at 6000 rpm for 5 minutes,and the supernatant was combined with the previously withdrawnsupernatant for the concentration measurement of unbound PEI. Theprecipitate was resuspended with 15 mL DD water as a stock solution forfurther uses.

3. Characterization of PEI-PAAIO

The evidence of successful PEI coating on PAAIO surface was confirmed byFourier transform infrared (FTIR). FTIR spectra were performed using aPerkin-Elmer system 2000 spectrometer. Dried samples were ground withpotassium bromide (KBr) powder and pressed into pellets for FTIRmeasurements. Sixty-four scans were signal-averaged in the range from4000 to 400 cm⁻¹ at a resolution of 4 cm⁻¹. Electron spectroscopy forchemical analysis (ESCA) measurements was performed on a ST PHI 5000Versa Probe (Sliedrecht, Netherlands) spectrometer using a monochromatedAl Kα X-ray source. The relative atomic concentration of each element atthe sample surface was calculated from the peak area using the atomicsensitivity factor. Spectra were recorded over a range of bindingenergies from 0 to 800 eV with a pass energy of 100 eV for the wide scansurvey and a pass energy of 20 eV for high energy resolution spectra forregions of N_(1s). The magnetic properties were measured using amagnetic properties measurement system (MPMS) from Quantum Design(MPMS-XL 7), which utilizes a superconducting quantum interferencedevice (SQUID) magnetometer at fields ranging from −15 to 15 K Oe at 25°C.

4. Preparation and Characterization of PEI-PAAIO/pDNA Magnetoplexes

Since the pDNA concentration in magnetoplexes was fixed, manipulatingthe PEI-PAAIO concentration could be used to adjust thenanoparticle/pDNA (N/P) ratios between non-viral gene vectors and theplasmid DNA. Equal volumes of the PEI-PAAIO and the pDNA solutions weremixed at N/P ratios from 1 to 30 and immediately vortexed at high speedfor 60 seconds. The PEI-PAAIO/pDNA magnetoplexes were kept at roomtemperature for 30 minutes for complete complexation before analysis.

DNA binding. DNA binding ability of magnetoplexes was evaluated byagarose gel electrophoresis. The magnetoplexes were prepared at variousN/P ratios using the procedure stated above. After 0.5 hour or 4 hoursstanding with or without 10% fetal bovine serum (FBS), the stability ofthe magnetoplexes was performed by gel electrophoresis with 0.8%agarose.

Dynamic light scattering (DLS) and zeta potential. The averagedhydrodynamic diameter and zeta potential of PAAIO, PEI-PAAIO andPEI-PAAIO/pDNA were measured by laser Doppler anemometry using aZetasizer Nano ZS instrument (Marlvern, Worcestershire, UK). Lightscattering measurements were carried out with a laser at 633 nm with a90° scattering angle. The concentration of the sample was 0.1 mg/mL andthe temperature was maintained at 25° C. Polystyrene nanospheres (220±6nm and −50 mV; Duke Scientific, USA) were used to verify the performanceof the instrument. The particle size and zeta potential of each samplewas performed in triplicate.

Transmission electron microscopy (TEM). The size and morphology ofmagnetoplexes were visualized by cryo-TEM (Jeol JEM-1200, Tokyo, Japan).A carbon coated 200 mesh copper specimen grid (Agar Scientific Ltd.Essex, UK) was glow-discharged for 1.5 minutes. One drop of the samplesolution was deposited on the grid and left to air-dry at roomtemperature, and was then examined with an electron microscope.

5. Cytotoxicity Assay

HEK 293T cells (human embryonic kidney 293T cell line) were seeded in12-well tissue culture plates at a density of 1×10⁵ cells per well inDulbecco's Modified Eagles' Medium (DMEM) containing 10% FBS for 24hours. Cytotoxicity of PEI-PAAIO (or PEI-PAAIO/pDNA magnetoplexes) wasevaluated by determining the cell viability after 4 hours incubation ina serum-free DMEM followed by 72 hours post incubation in the DMEMcontaining 10% FBS at the same condition. In addition, the cytotoxicityof the nanoparticle or magnetoplex-treated cells was examined in thepresence of an average 3000-G static magnetic field of Nd—Fe—B diskmagnets underneath the cells during the 4 hours incubation period. Thenumber of viable cells was determined by estimation of theirmitochondrial reductase activity using the tetrazolium-basedcolorimetric method (MTT conversion test).

6. Transfection Efficiency

HEK 293T cells were seeded at a density of 1×10⁵/well in 12 well platesand incubated in DMEM medium containing 10% FBS for 24 hours beforetransfection. When the cells were at 50% to 70% confluence, the culturemedium was replaced with 1 mL of DMEM with or without 10% FBS. Inaddition, magnetoplexes with 4 μg pEGFP-C1 (control) and PEI-PAAIO/pDNAmagnetoplex were prepared, and the medium was replaced with freshcomplete-medium and the cells were incubated for 72 hours posttransfection after pEGFP-C1 or PEI-PAAIO/pDNA magnetoplex was culturedwith cells for 4 hour incubation with or without a static magneticfield. The green fluorescent protein (GFP) expression was directlyvisualized under a fluorescence microscope.

For the luciferase assay, the procedures as stated above were repeatedto determine the transfection efficiency of the magnetoplexes comparedwith naked DNA (as a negative control) and PEI/pDNA polyplex at an N/Pratio of 10 (as a positive control) in HEK 293T cells and U87 (humanglioblastoma cell line) cells. To quantify the luciferase expression,transfected cells were rinsed gently with 1 mL of 0.1 M PBS (phosphatebuffered saline, twice), and then added to 200 μL/well of lysis buffer(0.1 M Tris-HCl, 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.1%Triton X-100, pH 7.8) and let stand overnight at −20° C. Next day, eachcell lysate was warmed to room temperature and centrifuged at high speedfor 30 minutes. The luciferase activity was monitored using the TopCountNXT™ microplate scintillation and luminescence counter (Perkin Elmer,N.J., USA) after mixing the supernatant with the luciferase assayreagent (Promega, Madison, Wis., USA). The total protein content of thecell lysate was examined using a BCA protein assay kit (Pierce Rockford,Ill., USA).

7. Cellular Uptake

The internalized iron amount of PEI-PAAIO was quantified by inductivelycoupled plasma-optical emission spectrometer (ICP-OES, Optima 7000DV,Perkin Elmer, Boston, Mass., USA). The protein content of the half celllysate was examined using the BCA protein assay kit, and the other halfcell lysate was dissolved in 37% HCl and incubated at 70° C. for 1 hour.The samples were diluted to a final volume of 3 mL for analysis. Theiron content of the samples were calculated based on an Fe(NO₃)₃calibration curve.

The internalized PEI-PAAIO nanoparticles within HEK 293T cells wasdirectly visualized by Prussian blue staining of iron. Prussian bluesolution was prepared by mixing 10 mL of 2% potassium hexacyanoferrate(II) trihydrate solution and 5 mL of 2% HCl. The internalized Fe³⁺ ionsof PAAIO turn to bright blue pigment when reacted with the ferrocyanideions. After 4 hours incubation with PEI-PAAIO as aforementioned, thecells were washed with 0.1M PBS (thrice) and fixed with 3.7%formaldehyde for 10 minutes, and washed again with PBS (thrice).Prussian blue solution of 1 mL/well was added and incubated with thecells for 30 minutes. Blue color can be visualized using an opticalmicroscope.

8. Statistical Methods

Means, standard deviation (SD), and standard error (SE) of the data werecalculated. Differences between the experimental groups and the controlgroups were tested using Student's-Newman-Keuls' test and p<0.05 wasconsidered significant.

Results 1. Characterization of PEI-PAAIO

Optimized conditions using various ratios between PEI and PAAIO to forma stable complex were tested using pH 2.0, 6.8 and 11. Two weight ratiosbetween PAAIO and PEI at 1:1 and 2:1 were chosen for particle size andzeta potential measurements. PAAIO had an average hydrodynamic diameterof 35 nm (FIG. 2( a)) and a negative zeta potential of −25 mV (FIG. 2(b)). Please continuously refer to FIG. 2( b), at pH 6.8, both PEI-PAAIOcomplexes showed similar zeta potentials of +22 mV but the PEI-PAAIOcomplex (PEI:PAAIO=1:2) had smaller particle diameter. Thus, thePEI-PAAIO complex (PEI:PAAIO=1:2) was chosen for all latter experiments.The positive zeta potential of the complexes explains the successfuldecoration of PEI on PAAIO. Please refer to the FTIR spectra of FIG. 3(a), PAAIO showed three characteristic stretchings at 1568, 1455 and 1406cm⁻¹ and PEI showed three characteristic stretchings at 1647, 1576 and1470 cm⁻¹. The primary amine stretching of PEI at 1647 cm⁻¹ shifted to1632 cm⁻¹ and the asymmetric carboxylate peak at 1568 cm⁻¹ shifted to1562 cm⁻¹ due to the electrostatic interactions. Moreover, the peakintensity of carboxylate stretching of PAAIO at 1562 cm⁻¹ decreasedbecause of a PAAIO surface blocking by PEI. The further evidence of PEIdecorated on PAAIO was also observed by ESCA where the binding energiesof atoms C_(1s), N_(1s) and O_(1s) appear at 283, 400 and 530 Ev (FIGS.3( b) and 3(c)). The atom percentage of N was undetectable in PAAIO butthe value of 4.7% was measured in PEI-PAAIO complex. The saturationmagnetization (Ms) value of PEI-PAAIO complex was 76 emu/g Fe and 103emu/g Fe for PAAIO (FIG. 3( d)). The Ms decreased after PEI coated onPAAIO. PEI-PAAIO complex remained superparamagnetic at room temperatureand showed negligible hysteresis.

The concentration of PEI in PEI-PAAIO was determined by measuring thecuprammonium complex formed between PEI and copper (II) at 630 nm usinga UV-vis spectrophotometer (Namgung et al., 2010). The value of0.135±0.03 mg/mL was obtained based on a calibration curve of PEI. Whenthe molecular weights of PAAIO and PEI were 2232 g/mol and 25,000 g/molrespectively, the molar percent of PEI in PEI-PAAIO was 1.37 mol %. Thisvalue was utilized to calculate N/P ratios between PEI-PAAIO and pDNA.The theoretical PEI value in feed was 33 wt %. The theoretical molarratio between the amino units in PEI and the carboxylate units in PAAwas approximately 3 to 1 in feed. The 13.5 wt % of PEI could becorrelative to the ratio of 1.25 to 1 between amino and carboxylategroups.

2. Characterization of PEI-PAAIO/pDNA Magnetoplexes

By changing PEI-PAAIO weight and keeping pDNA weight as a constant,various N/P ratios of PEI-PAAIO/pDNA magnetoplexes were prepared. Thebinding ability between PEI-PAAIO and pDNA was studied by agarose gelelectrophoresis retardation assay. The result shows that the pDNA couldbe well encapsulated at an N/P ratio higher than 5, and a wellprotection of pDNA from FBS degradation was observed at an N/P ratio ofhigher than 15. Therefore, it was ensured that pDNA within PEI-PAAIO wasnot degraded with serum and the stability of pDNA was maintained, and 4hours incubation condition was chosen for the latter transgeneexpression.

Please refer to FIG. 4( a), the hydrodynamic diameters of PAAIO andPEI-PAAIO were 30 nm and 50 nm measured by DLS. The particle averagediameter of PEI-PAAIO/pDNA magnetoplexes increased to 150 nm at N/Pratios of 3 and 5, maximized at 7 (250 nm) and decreased dramatically toless than 100 nm when an N/P ratio was larger than 9. Please refer toFIG. 4( b), the surface charge of PAAIO is −30 mV and turns to apositive value of 16 mV following PEI decoration. This positive value ofzeta potential also ascertains the successful coating of PEI onto PAAIO.The PEI-PAAIO/pDNA magnetoplexes formed from a low amount of PEI-PAAIOshowed negative surface charges (at the N/P ratios of 3 and 5). Themagnetoplex at the N/P ratio of 7 displayed a fluctuation of zetapotential between positive and negative values. The serious fluctuationin zeta potential may be attributed to a charge balance between positivePEI-PAAIO and negative pDNA at this N/P ratio. Increase in an N/P ratioof larger than 7 resulted in an increase in positive zeta potential ofmagnetoplexes.

The TEM morphological image of PAAIO shows an average particle diameterof 8.62±1.82 nm. The average particle diameter of PEI-PAAIO decorated byPEI slightly increased to 9.31±3.21 nm. All PEI-PAAIO/pDNA magnetoplexesat N/P ratios of 15, 20, and 30 appeared as clusters with evenlydistributed bare iron oxide particles, and the measured cluster particlediameters were 103.6±16.5, 105.4±19.9, 128.8±14.4 and 95.8±11.2 nm inorder. Larger sized MNP has been reported to exhibit higher transfectionrates compared with small sized MNP (Chorny et al., 2007). Thus, thelarger diameter of PEI-PAAIO/pDNA than that of the MNP may have thebetter magnetofection.

3. Cell Viability

The cell viability of PEI-PAAIO was tested by MTT assay using HEK 293Tcells as stated above. Please refer to FIG. 5( a), PEI-PAAIO exhibits nocytotoxicity in all tested concentrations. The high cell viability ofPEI-PAAIO may be attributed to the low PEI content (The percentage ofPEI was only 13.5 wt % in PEI-PAAIO). Please refer to FIG. 5( b),PEI-PAAIO/pDNA magnetoplexes with an N/P ratio ranging from 1 to 30 alsoshowed minimal cytotoxicity. For instance, the PEI concentration inPEI-PAAIO at an N/P ratio of 30 was 16.2 μg/mL, where HEK 293T cellsstill remained 80% viable. The cytotoxicity of the PEI-PAAIO/pDNAmagnetoplex at N/P ratio of 30 was dramatically less than PEI/pDNApolyplex prepared at an N/P ratio of 10. Thus, the PEI-PAAIO can be usedas a non-viral gene vector superior to pure PEI if the cytotoxicity ofgene vectors is a major concern. In addition, the external magneticfield applied underneath the cell culture plate does not influence thecell viability.

4. In Vitro Gene Transfection

The aforementioned experiments have demonstrated that PEI-PAAIO endowsthe good condensation ability with pDNA and remains minimalcytotoxicity. As the experiment “6. Transfection efficiency” as above,two plasmid DNAs were used to test the transfection efficiency ofPEI-PAAIO as a non-viral gene carrier, in which plasmids pEGFP-C1 andpGL3-control were chosen respectively for fluorescence and luminescencemeasurements.

The relative green fluorescence expression from pEGFP-C1 was traced byfluorescence microscopy. FIG. 6( a) and FIG. 6( b) respectively showedthe relative green fluorescence at the conditions without 10% FBS andwith 10% FBS. Please refer to FIG. 6( a), the transfection efficiencyincreased with an increase in the N/P ratio between PEI-PAAIO andpEGFP-C1 and reached the maximum at the N/P ratio of 25. Upon applying amagnetic field, the transfection efficiency was improved at the N/Pratios of 15 and 20 but decreased at the ratios of 25 and 30. Pleaserefer to FIG. 6( b), the green fluorescence intensity of PEI/pDNAreduced dramatically when 10% FBS was added to the cell culture medium.This may be due to the competition between FBS and pDNA with PEI, whichreduces the pDNA concentration in the magnetoplexes. In addition, theFBS adsorption on PEI/pDNA surface has also been reported to inhibittransgene expression. Screening of transfection efficiencies undervaried N/P ratios (PEI-PAAIO:pEGFP) without FBS revealed no magneticenhancement of gene expression when the N/P ratio was less than 9. Oncontrast, a remarkable magnetic-enhanced effect was observed for thePEI-PAAIO/pEGFP magnetoplexes at the N/P ratio of larger than 15. Thiscan be reasoned that the rapid internalization of the magnetoplexes withthe high N/P ratios into the cells prevents pDNA destabilization fromFBS interference during that incubation period.

Next, a quantitative comparison of transfection ability of themagnetoplexes with the PEI-PAAIO polyplex with and without 10% FBS wasalso conducted by measuring luciferase gene expression usingpGL3-control plasmid. Please refer to FIGS. 7( a) and 7(b), theconsistent result revealed that in the presence or absence of FBS, asignificantly improved transgene expression was obtained when a magneticfield was utilized to pull the PEI-PAAIO/pDNA magnetoplexes into thecells.

5. Cellular Uptake

The cellular uptake of PEI-PAAIO in HEK 293T cells was directly stainedby Prussian blue. No discrete difference is shown with and without animposed magnetic field in the absence of 10% FBS. Nevertheless, in thepresence of 10% FBS, the blue color image becomes clearer with anincreasing N/P ratio between PEI-PAAIO and pDNA under a magnetic field.It is demonstrated in the present invention that the larger particlediameters of PEI-PAAIO/pDNA magnetoplexes clearly demonstrated a highercellular internalization when a magnetic field was applied.

In addition, the quantity of iron normalized to total cell populationwas determined by ICP-OES. Please refer to FIGS. 8( a) and 8(b), whichrespectively illustrate the quantity of iron within cells (a) without10% FBS and (b) with 10% FBS in the absence or presence of a magneticfield. The internalized iron amount within the cells showed nocorrelation with an increasing N/P ratio when a magnetic field wasabsent. On the contrary, the cellular uptake of iron oxide particlesincreased with an increasing N/P ratio under a magnetic field.Independent of FBS, the internalized iron oxide particles increased uponan imposed magnetic field. At an N/P ratio of 30, the internalized ironamount was approximately 2-fold if the magnetic field was applied. Insummary, PEI-PAAIO/pDNA magnetoplexes is very efficient for genetranfection in HEK 293T cells in a circumstances of FBS.

Please refer to FIGS. 9( a) and 9(b), which respectively illustrate themagnetic transfection of U87 cells (a) without 10% FBS and (b) with 10%FBS in the absence or presence of a magnetic field, and the transfectionefficiency increases with an increase in the N/P ratio. In FIG. 9( b),the stimulation by the magnetic field still augments the transfectionefficiency significantly. At an N/P ratio of larger than 7, thePEI-PAAIO/pDNA magnetoplexes showed higher transfection efficiency thanPEI/pDNA polyplexes. Please refer to FIGS. 9( c) and 9(d), whichrespectively illustrate the internalized iron amount within U87 cellsmeasured by ICP-OES (c) without FBS and (d) with FBS. Similarly, withoutFBS, no detectable variation in the internalized iron amount was foundbetween with and without magnetic force. Nevertheless, in the FBScondition, U87 cells showed significant improvement in the amount ofinternalized iron and gene transfection in comparison with 293T cellswhen a magnetic field was present.

6. Conclusion

In the present invention, PAAIO was successfully coated withpolyethylenimine (PEI) to form the PEI-PAAIO polyplex, and plasmid DNAthen was absorbed on the PEI via electrostatic coupling. The stable andsuperparamagnetic PEI-PAAIO/pDNA magnetoplex was formed, which had abetter transfection efficiency than PEI, and thus PEI-PAAIO could be theexcellent non-viral gene vector. Under an applied magnetic field, thegene transfection efficiency of the magnetoplexes of the presentinvention was enhanced especially in the presence of FBS. The resistanceto disruption from serum proteins is a benefit of using thePEI-PAAIO/pDNA magnetoplex for clinical applications, such as genetherapy or the powerful gene transfection tool.

EMBODIMENTS

1. A method for preparing a nanoparticle, including steps of: reacting apolyethylenimine (PEI) with a polyacrylic acid-bound iron oxide (PAAIO)to obtain a polyelectrolyte complex (PEC); and incorporating the PECwith a genetic material to form the nanoparticle.

2. The method according to embodiment 1, wherein the PAAIO is obtainedby reacting a polyacrylic acid with an iron oxide.

3. The method according to at least one of embodiments 1 and 2, whereinthe nanoparticle is a magnetic nanoparticle.

4. The method according to at least one of embodiments 1 to 3, whereinthe PEC further includes the bound PEI, and the unbound PEI is removedby a magnetic force.

5. The method according to at least one of embodiments 1 to 4, whereinthe genetic material is one selected from a group consisting of a DNA,an RNA, a complementary DNA, a micro RNA and a small interfering RNA.

6. The method according to at least one of embodiments 1 to 5, whereinthe PAAIO is water soluble, and the nanoparticle has a superparamagneticproperty.

7. A nanoparticle, including: a PEC including a PEI and a PAAIOconfigured on the PEI; and a genetic material coupled to the PEI.

8. The nanoparticle according to embodiment 7, wherein the geneticmaterial is one selected from a group consisting of a DNA, an RNA, acomplementary DNA, a micro RNA and a small interfering RNA.

9. The nanoparticle according to at least one of embodiments 7 to 8,wherein the PAAIO is water soluble.

10. The nanoparticle according to at least one of embodiments 7 to 9,wherein the PEC has a first concentration and a first volume, thegenetic material has a second concentration and a second volume, the PECand the genetic material are dissolved in a water, the first volume isequal to the second volume, and the first concentration and the secondconcentration have a ratio ranged from 1/1 to 1/30.

11. A nanoparticle, including: a PEC including a positive chargemolecule and a magnetic particle configured on the positive chargemolecule; and a genetic material coupled to the positive chargemolecule.

12. The nanoparticle according to embodiment 11, wherein the positivecharge molecule is one selected from a group consisting of a PEI and aPEGlyated PEI.

13. The nanoparticle according to at least one of embodiments 11 to 12,wherein the metal particle is water soluble and is one selected from agroup consisting of a gold nanoparticle, a silver nanoparticle and aniron nanoparticle.

14. The nanoparticle according to at least one of embodiments 11 to 13,wherein the genetic material is one selected from a group consisting ofa DNA, an RNA, a complementary DNA, a micro RNA and a small interferingRNA.

15. The nanoparticle according to at least one of embodiments 11 to 14,wherein the magnetic particle is a metal particle.

16. The nanoparticle according to at least one of embodiments 11 to 15,wherein the genetic material is electrostatically coupled to thepositive charge molecule.

While the invention has been described in terms of what is presentlyconsidered to be the most practical and preferred Embodiments, it is tobe understood that the invention needs not be limited to the disclosedEmbodiments. On the contrary, it is intended to cover variousmodifications and similar arrangements included within the spirit andscope of the appended claims, which are to be accorded with the broadestinterpretation so as to encompass all such modifications and similarstructures.

1. A method for preparing a nanoparticle, comprising steps of: reactinga polyethylenimine (PEI) with a polyacrylic acid-bound iron oxide(PAAIO) to obtain a polyelectrolyte complex (PEC); and incorporating thePEC with a genetic material to form the nanoparticle.
 2. The methodaccording to claim 1, wherein the PAAIO is obtained by reacting apolyacrylic acid with an iron oxide.
 3. The method according to claim 1,wherein the nanoparticle is a magnetic nanoparticle.
 4. The methodaccording to claim 3, wherein the PEC comprises the bound PEI, and theunbound PEI is removed by a magnetic force.
 5. The method according toclaim 1, wherein the genetic material is one selected from a groupconsisting of a DNA, an RNA, a complementary DNA, a micro RNA and asmall interfering RNA.
 6. The method according to claim 1, wherein thePAAIO is water soluble, and the nanoparticle has a superparamagneticproperty.
 7. A nanoparticle, comprising: a polyelectrolyte complex (PEC)comprising a polyethylenimine (PEI) and a polyacrylic acid-bound ironoxide (PAAIO) configured on the PEI; and a genetic material coupled tothe PEI.
 8. The nanoparticle according to claim 7, wherein the geneticmaterial is one selected from a group consisting of a DNA, an RNA, acomplementary DNA, a micro RNA and a small interfering RNA.
 9. Thenanoparticle according to claim 7, wherein the PAAIO is water soluble.10. The nanoparticle according to claim 7, wherein the PEC has a firstconcentration and a first volume, the genetic material has a secondconcentration and a second volume, the PEC and the genetic material aredissolved in a water, the first volume is equal to the second volume,and the first concentration and the second concentration have a ratioranged from 1/1 to 1/30.
 11. A nanoparticle, comprising: apolyelectrolyte complex (PEC) comprising a positive charge molecule anda magnetic particle configured on the positive charge molecule; and agenetic material coupled to the positive charge molecule.
 12. Thenanoparticle according to claim 11, wherein the positive charge moleculeis one selected from a group consisting of a polyethylenimine (PEI) anda PEGlyated PEI.
 13. The nanoparticle according to claim 11, wherein themetal particle is water soluble and is one selected from a groupconsisting of a gold nanoparticle, a silver nanoparticle and an ironnanoparticle.
 14. The nanoparticle according to claim 11, wherein thegenetic material is one selected from a group consisting of a DNA, anRNA, a complementary DNA, a micro RNA and a small interfering RNA. 15.The nanoparticle according to claim 11, wherein the magnetic particle isa metal particle.
 16. The nanoparticle according to claim 11, whereinthe genetic material is electrostatically coupled to the positive chargemolecule.